Padlock Probe-Based Assays for

Molecular Diagnostics

Anja Mezger

©Anja Mezger, Stockholm University 2015

ISBN 978-91-7649-155-3 pp1-53

Printed in Sweden by Holmbergs, Malmö 2015

Distributor: Department of Biochemistry and Biophysics,

Stockholm University

The possibilities are limited only

by our imagination and determination,

and not by the physics.

Mike Duke

To my family,

for believing in me

List of Publications

This thesis is based on the following papers:

Mezger A, Öhrmalm C, Herthnek D, Blomberg J, Nilsson M. Detection of

rotavirus using padlock probes and rolling circle amplification. PLoS One 9,

e111874 (2014).

Mezger A, Gullberg E, Göransson J, Zorzet A, Herthnek D, Tano E, Nilsson

M*, Andersson DI*. A general method for rapid determination of antibiotic

susceptibility and species in bacterial infections. J Clin Microbiol 53, 425-432

(2015).

Mezger A, Allen S, Cavelier L, Hultén M, Nilsson M. Elimination of maternal

DNA for accurate non-invasive prenatal testing: a pilot study. Submitted.

Mignardi M*, Mezger A*, Larsson C and Nilsson M. Oligonucleotide gap-fill

ligation for mutation detection and sequencing in situ. Submitted – under review.

* These authors contributed equally.

Reprints were made with permission from the publishers.

Related work by the author

Mezger A*, Kühnemund M*, Nilsson M, Herthnek D. Highly specific DNA

detection employing ligation on suspension bead array readout. New

Biotechnology (2015). [Epub ahead of print].

Østerberg FW, Rizzi G, Donolato M, Bejhed RS, Mezger A, Strömberg M,

Nilsson M, Strømme M, Svedlindh P, Hansen MF. On-chip detection of rolling

circle amplified DNA molecules from Bacillus globigii spores and Vibrio

cholerae. Small 10, 2877-2882 (2014).

Gomez de la Torre TZ*, Ke R*, Mezger A, Svedlindh P, Strømme M, Nilsson

M. Sensitive detection of spores using volume-amplified magnetic nanobeads.

Small 8, 2174-2177 (2012).

Gomez de la Torre TZ, Mezger A, Herthnek D, Johansson C, Svedlindh P,

Nilsson M, Strømme M. Detection of rolling circle amplified DNA molecules

using probe-tagged magnetic nanobeads in a portable AC susceptometer.

Biosens Bioelectron 29, 195-199 (2011).

* These authors contributed equally.

Contents

Introduction ............................................................................................ 9

In vitro nucleic acid detection ............................................................... 10

Polymerization-based technologies ......................................................................10

Ligation-based technologies ................................................................................12

Sequencing-based technologies ...........................................................................14

In situ nucleic acid detection................................................................. 17

Fluorescent in situ hybridization ..........................................................................17

Methods for signal amplification in situ .................................................................18

In situ sequencing .............................................................................................20

Diagnostic methods used in bacteriology and virology .......................... 21

Bacterial identification by culture and biochemical methods ....................................21

Microscopy for rapid microbial diagnosis ...............................................................22

Immunoassays ..................................................................................................23

PCR-based methods for pathogen identification .....................................................23

Mass spectrometry ............................................................................................24

Methods for antibiotic susceptibility testing ...........................................................25

Summary of diagnostic methods .........................................................................27

Methods for prenatal diagnosis of chromosomal aneuploidies .............. 28

Traditional prenatal testing .................................................................................28

Non-invasive prenatal testing ..............................................................................29

Molecular Diagnostics in Oncology ........................................................ 32

Present investigations ........................................................................... 34

Detection of rotavirus using padlock probes and rolling circle amplification ...............34

A general method for rapid determination of antibiotic susceptibility and species in

bacterial infections ............................................................................................35

Elimination of maternal DNA for accurate non-invasive prenatal testing: a pilot study 36

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ ...........37

Populärvetenskaplig sammanfattning på svenska ................................. 40

Acknowledgments ................................................................................. 41

References ............................................................................................ 44

Abbreviations

ACTB Actin beta

ASP Antibiotic susceptibility profile

AST Antibiotic susceptibility testing bDNA Branched DNA C2CA Circle-to-circle amplification

cff-DNA Cell-free fetal DNA

CLIA Clinical Laboratory Improvement Amendments

CVS Chorionic villus sampling

EGFR Epidermal growth factor receptor

EIA Enzyme immunoassay

ELISA Enzyme-linked immunosorbent assay

EM Electron microscopy

FISH Fluorescent in situ hybridization

FRET Fluorescence resonance energy transfer

HER2 Human epidermal growth factor receptor 2

HIV Human immunodeficiency virus

ISH In situ hybridization

KRAS Kirsten rat sarcoma viral oncogene homolog

LAMP Loop-mediated isothermal amplification

LNA Locked nucleic acid

MALDI Matrix-assisted laser desorption/ionization

MELAS Mitochondrial encephalomyopathy, lactic

acidosis, and stroke-like episodes

MIC Minimum inhibitory concentration

MLPA Multiplex ligation-dependent probe amplification

MPS Massively parallel sequencing

MS Mass spectrometry

MSAFP Maternal serum alpha-fetoprotein

NGS Next-generation sequencing

NIPD Non-invasive prenatal diagnosis

NIPT Non-invasive prenatal testing

OLA Oligonucleotide ligation assay

PAPP-A Pregnancy-associated plasma protein-A

PCR Polymerase chain reaction

PNA Peptide nucleic acid

QF-PCR Quantitative fluorescent PCR

qPCR Quantitative PCR

RCA Rolling circle amplification

RCP Rolling circle product

RT-PCR Reverse-transcription PCR

SDA Strand displacement amplification

smFISH Single-molecule FISH

SNV Single nucleotide variant

TB Tuberculosis

TOF Time of flight

UTI Urinary tract infection

WHO World Health Organization

β-hCG β-human chorionic gonadotropin

9

Introduction

Rapid and reliable identification of the factors underlying a disease is essential

for correct treatment and ultimately for the patient’s health. The optimal test

should excel in several parameters in order to guarantee accurate and affordable

diagnosis. Clinical and analytical sensitivity must be high, as well as specificity

and precision. Especially in infectious disease diagnosis, an assay must be rapid

in order to minimize the time until adequate treatment can be initiated.

Additionally, cost, required technical skills and infrastructure must be kept to a

minimum to allow wide application, also in low resource settings. A plethora of

diagnostic tools already exists, but none of them excels in all stated aspects.

Although it should be noted, that the importance of each parameter varies from

field to field. Time, for example, is not as important in oncology and prenatal

testing as it is in infectious disease diagnostics.

The ability of delivering effective care depends on accurate diagnosis.

Molecular assays have the advantage of providing additional information on the

nucleic acid or protein level, which can be used to maximize the clinical benefit.

Knowledge of the nucleic acid content allows not only rapid diagnostics, but

also targeted therapies. The work presented in my thesis focused on the

development of nucleic acid-based methods for molecular diagnostics. The aim

was to develop several assays that have the potential to be applied in clinical

practice as they overcome certain limitations of already existing methods. The

covered areas include viral detection, antibiotic resistance profiling, non-

invasive prenatal diagnosis (NIPD) and in situ mutation detection,

demonstrating the wide applicability of padlock probes for clinical use.

In this thesis, I will first give an overview of molecular technologies used for

nucleic acid detection, which are the basis of a range of diagnostic tests. Then, I

will describe nucleic acid and protein-based technologies that are commonly

applied in clinical diagnostics. The advantages of nucleic acid-based diagnostics

in the fields of infectious disease diagnostics, prenatal testing and in oncology

will be discussed in the respective subsection. In the last part, I will describe and

discuss the developed assays that this thesis is composited of and give a future

outlook.

10

In vitro nucleic acid detection

A number of methods are currently available for nucleic acid detection aiming at

identifying a specific nucleic acid sequence. This goal can be reached by

techniques that are mainly based on polymerization, ligation, sequencing or a

combination of these. I will give an overview of some of the most prominent

techniques on which many assays, currently used in the field of molecular

diagnostics, are based.

Polymerization-based technologies

Polymerization can be used to specifically detect and amplify a target sequence

as polymerases possess the enzymatic ability to copy the sequence content of a

template strand. The most widely used polymerization-based amplification

technique is the polymerase chain reaction (PCR). Invented 30 years ago it

allows the exponential amplification of a target sequence1-3

. The target DNA

sequence is amplified by cyclically heat denaturing the DNA double strand,

annealing the two primers and extending them by polymerization. The primers

hybridize to complementary sequences of the target strand and thus, flanking the

target site. Thermal cycling is required since the temperature must be repeatedly

altered between the denaturation, extension and annealing steps. The

development of quantitative PCR (qPCR) made accurate quantification of the

target DNA possible. In real-time qPCR, the amplification process is

continuously monitored by measuring the increase in fluorescence whereby the

fluorescence intensity is directly proportional to the target DNA concentration

and can be used for DNA quantification4, 5

. Dyes, such as SYBR green I that

preferentially stain double stranded DNA or alternatively, fluorescent resonance

energy transfer (FRET) probes can be used. FRET probes are short

oligonucleotide probes labeled with a fluorophore/quencher pair. The most

commonly used FRET probes are TaqMan probes. Upon binding of the probe

and subsequent hydrolysis by the 5’-3’ exonuclease activity of the Taq

polymerase the fluorophore is released and due to the lack of proximity it is no

longer quenched4, 6, 7

. qPCR can accurately detect a two-fold difference in DNA

concentration8. Absolute quantification can be achieved by the inclusion of an

internal standard9 or by the use of a standard curve that is constructed by

amplification of known amounts of target nucleic acids10

. Additionally, qPCR

offers a lower risk of cross-contamination as a closed system from sample to

readout can be used. Furthermore, the turnaround time is significantly

decreased, since quantification does not require any post-PCR manipulation.

Digital PCR achieves an even higher quantitative accuracy whereby the target

DNA is diluted and compartmentalized with a concentration of less than one

11

copy per compartment and individually amplified11

. The number of positive

wells is counted after amplification reflecting the number of initial target

molecules present. Digital PCR allows the analysis of rare mutations11-13

, which

has been difficult with conventional PCR in which bulk DNA, compared to

single copies, is analyzed. Increasing the number of compartments leads to an

increase in sensitivity as a larger fraction of the sample is analyzed11, 14

. The

introduction of droplet microfluidics allows the analysis of a large number of

compartments in a cost-effective manner without the need for larger sample

volumes15

.

All PCR-based methods require precise temperature control. Isothermal

amplification methods, on the other hand, do not rely on thermal cycling and

thus implementation into microfluidic chips and use in low resource settings is

facilitated. Several polymerization-based isothermal amplification methods have

been developed in the last decades. Two isothermal amplification techniques,

both used in diagnosis of infectious diseases16-19

, are described in greater detail

below, followed by a description of rolling circle amplification (RCA).

Strand displacement amplification (SDA) uses primers containing a recognition

site for a nicking enzyme and a target complementary part. After initial

extension of the primers, the nicking enzyme creates a free 3’ end, which is

extended by an exonuclease-deficient polymerase displacing the downstream

strand. Exponential amplification of the target sequence can be achieved by

using two primers targeting strands of opposite polarities20

. The target site must

not include the recognition site of the enzyme used as it would be digested by

the nicking enzyme during the amplification process. Another exponential

isothermal amplification method that has been used for detection of

microorganisms is the loop-mediated isothermal amplification (LAMP)

method18, 19

. LAMP requires several primers and a polymerase possessing

strand-displacement activity21

. The amplification product contains stem-loop

structures, which will initiate further amplification. A disadvantage of LAMP is

the complicated primer design, targeting six different genomic regions using

four primers21

. The likelihood of primer-primer interactions becomes larger with

the increase in the number of primers and thus, makes multiplexing difficult22

.

The work in my thesis is based on RCA, which is an efficient and well-exploited

isothermal method to amplify short DNA circles, including padlock probes

(described in more detail below)23-25

. A short primer hybridized to the circle

initiates polymerization. φ29 DNA polymerase possesses several characteristics

that make it an excellent choice for RCA. It is a highly processive enzyme and

does not require accessory proteins26

. Furthermore, the strand displacement

activity enables a continuous amplification of the circularized padlock probe26

and the 3’ to 5’ exonuclease activity allows to efficiently use the DNA target as

a primer by digesting overhanging nucleotides (target-primed RCA)27

.

12

Common for all mentioned methods is the fact that specificity solely depends on

primer hybridization. An increase in specificity can be achieved by using the

intrinsic properties of ligases to distinguish single base mismatches.

Ligation-based technologies

In nature, ligation is used during replication and as a repair mechanism to seal

single-strand breaks in duplex DNA or to repair double-strand breaks28-31

.

Ligases catalyze the formation of a phosphodiester bond between the 3’

hydroxyl group of one DNA strand and the 5’ phosphate group of the other

DNA strand30, 31

. The ligation of single-strand breaks is templated by the

complementary strand and mismatches at the ligation site inhibit strand sealing

to some extent with the highest discriminatory power at the 3’ end of the nick32-

35. Several DNA detection and amplification technologies are based on the

above described properties of ligases. The oligonucleotide ligation assay (OLA)

was one of the first technologies that utilized the specificity of DNA ligases36

.

Although not a nucleic acid amplification method, I will shortly describe OLA

as several other methods are based on its principle. The first description of the

method used T4 DNA ligase to ligate two adjacent complementary

oligonucleotides and thereby discriminating single nucleotide mismatches36

. A

ligation product is only formed if the two oligonucleotides are perfectly base-

paired at the ligation site. By using a thermostable ligase and thermal cycling the

ligation product can be linearly amplified (termed ligase detection reaction)37

.

The ligation product is first heat-denatured from its target sequence and by

subsequent lowering of the temperature two new short oligonucleotides can

hybridize and be ligated and thus be linearly amplified37

. Addition of a second

set of oligonucleotides, complementary to the first one, results in exponential

amplification (termed ligase chain reaction)37

.

Another example of a ligation-based amplification method, which has been

commercialized for mutation detection in various genetic disorders (MRC-

Holland), is the multiplex ligation-dependent probe amplification (MLPA)38

. In

MLPA, two probes, built up of identical end sequences for primer binding and a

target complementary part that, like in OLA, hybridize to adjacent target sites39

.

Spacer sequences of different length between the primer binding and the target

site allow multiplex detection and amplification39

. Upon ligation the probes are

amplified by PCR and separated by capillary electrophoresis39

.

Padlock probes, on which the work in this thesis is based on, are a further

development of OLA. Padlock probes are linear oligonucleotides with two target

complementary arms that are linked via a backbone sequence (Figure 1)40

. The

backbone can contain sequences used for detection or recognition sites for

restriction enzymes. Upon hybridization the two ends are enzymatically joined

13

by ligation forming a topologically locked circle40

. Like in OLA specificity

relies on hybridization and on the fidelity of the ligase32, 36, 40

. Padlock probes are

locally amplified by RCA, by a factor of 1,000 per hour25

, and collapse

spontaneously into µm-sized coiled structures25, 41

. These amplification products,

termed rolling circle products (RCPs) can either be directly detected25

or further

amplified by, for example, circle-to-circle amplification (C2CA)42

. RCPs can be

detected in numerous ways, such as fluorescence based microscopy yielding a

digital assay43

or by colorimetric methods44

. C2CA increases sensitivity and

allows detection of target sequences in a wide dynamic range42, 43

. To initiate a

second round of RCA, the RCPs must first be monomerized. A short

oligonucleotide, containing an enzymatic restriction site, is hybridized to its

complementary sequence in the RCP and digested upon addition of a restriction

enzyme. These short monomers are re-ligated to form circles, which in turn can

be amplified by RCA42

. The use of padlock probes compared to the

amplification techniques described above offers several advantages: (i) single

nucleotide resolution can be achieved due to the high fidelity of the ligase45, 46

,

(ii) padlock probes can be highly multiplexed without the need of extensive

optimization allowing the simultaneous detection and amplification of more than

10,000 target sites47

, (iii) the first round of RCA products are topologically

linked to their target sequence40

allowing washing steps and yielding spatial

information if applied in situ. The latter will be discussed in a later section( in

situ RCA and sequencing).

14

Figure 1. Schematic illustration of padlock probes and rolling circle amplification. A) Illustration of a padlock

probe. The two target complementary arms are linked by a backbone sequence, which can contain different

sequence elements such as a restriction site (in blue) and a site for hybridization of a detection oligonucleotide

(in green). B) A ligase (in pink) seals the nick of the two juxtaposed padlock probe arms. C) The circular

molecule is amplified by φ29 DNA polymerase (in yellow) creating a concatemer of multiple copies of a

sequence with opposite polarity to the padlock probe. The resulting rolling circle product can be monomerized

by enzymatic digestion after hybridization of a restriction oligonucleotide to the restriction site (not shown). D)

The monomers are re-ligated, templated by undigested restriction oligonucleotides. E) Ligated circles are

amplified in a second amplification step. The amplified products can be detected in an optical imaging system by

hybridization of short fluorescently labeled oligonucleotides (not shown).

Sequencing-based technologies

DNA sequencing technologies have been implemented into diagnostics, as the

sequence content may give valuable information about disease and thus, allow

administration of effective treatment. Application areas include mutation

detection in cancer and hereditary diseases, prenatal testing and microbiology

15

where sequencing is mainly used for epidemiological purposes. With the

introduction of next-generation sequencers, sequencing costs have dropped

significantly and this trend is expected to continue48

, therefore sequencing might

become the diagnostic tool.

In 1977, one of the first sequencing methods was developed: Sanger

sequencing49

. For the next three decades, until the next-generation sequencing

(NGS) technologies arose, Sanger sequencing was practically the only

technology used in any sequencing project50

. In Sanger sequencing, the four

nucleotides are mixed together with their dideoxynucleotide analogs, which can

be incorporated into a DNA sequence but cannot be elongated, creating a

mixture of sequence fragments of different lengths49

. The original DNA

sequence can be derived after separating the resulting fragments according to

their length49

. Sanger sequencing excels at read length but suffers high costs.

Although offering long read lengths, this sequencing method is tedious and

unsuitable for high throughput. Substantial progress was made with the

introduction of an automated DNA sequencer using fluorescently labeled

primers and thus, eliminating the need of X-ray film development51

. Further

development of the sequencing chemistry included the introduction of

fluorescently labeled dideoxynucleotides permitting the synthesis of

fluorescence-tagged fragments in one reaction52

. The introduction of capillary

array electrophoresis for separation and detection of sequencing products

substantially increased throughput53

and thereby significantly contributed to the

completion of the human genome project54

.

In 2005, the breakthrough came with the introduction of two novel NGS

technologies: the 454 sequencing-by-synthesis technology55

and the multiplex

polony sequencing technology, based on sequencing-by-ligation56

. The 454

technology uses a modified version of the pyrosequencing protocol to sequence

the template strand which is coupled to beads in picolitre-sized reaction wells55

.

Signal detection is based on the detection of pyrophosphate, released during

nucleotide incorporation57

. Polony sequencing, developed by Shendure et al., on

which the sequencing by oligonucleotide ligation and detection (SOLiD)

technology is based, uses beads that bind the amplification products generated

by emulsion PCR56

. Fluorescently labeled degenerated probes are hybridized to

the amplification products and ligated to an anchor primer (Figure 2). Multiple

cycles of hybridization, ligation, detection and cleavage are performed whereby

the read length is determined by the number of cycles56

. Besides the two NGS

technologies mentioned above several alternative technologies have been

developed in the last decade. Illumina uses single-nucleotide addition of

reversible dye terminators, each nucleotide being identified by a specific

fluorescent label58

. Ion Torrent, on the other hand, does not use fluorescence to

determine the sequence content, but measures the pH change caused by the

release of hydrogen ions during nucleotide incorporation59

. The described NGS

16

technologies rely all on the same basic principles. First, a sequence library of

fragments with covalently attached adaptors is constructed to form clusters of

the original templates. Second, the sequencing is massively parallel, consisting

of a nucleotide addition, detection and a cleaving/washing step. As a

consequence, NGS technologies allow higher throughput at a fraction of the cost

required for Sanger sequencing. The downside is the shorter read length, which

makes it more difficult to uniquely map the reads to a reference genome. The

read length is determined by the signal-to-noise ratio, averaging 50-150 bp for

most NGS compared to 800-900 bp achieved with Sanger sequencing60

.

Figure 2. Schematic illustration of sequencing-by-ligation chemistry. An anchor primer is hybridized to its

target sequence and ligated to fluorescently labeled, degenerated nonamers. After image acquisition the ligation

product is stripped off and the cycle is repeated.

Sequencing of individual molecules, opposed to sequencing clusters, has the

advantage of eliminating the bias produced during library preparation and the

possibility of detecting DNA modifications, such as methylation. The difficulty,

though, is signal detection, as the sensor needs to be sensitive enough to detect

the signal changes from a single molecule. Pacific Biosciences, commercialized

in 2010, uses a technology where library fragments are bound to a single DNA

polymerase molecule, which is then deposited on the chip surface61

. The

polymerase has been engineered to allow incorporation of fluorescently labeled

nucleotides and the polymerization rate has been decreased in order to detect

single incorporated nucleotides61

. In 2012, sequencing of DNA methylations has

been achieved using the technology developed by Pacific Biosciences62, 63

. An

alternative to the above described single molecule NGS technology is the use of

nanopores. Nanopores function as ion channels and measure the change in ion

flux when a single DNA molecule is threaded through the pore64

. The four

nucleotides can be distinguished due to their unique electrical signature65

.

However, a major challenge remains to slow down the translocation rate as

commercial detectors lack sufficient temporal resolution causing a high error

rate66

. Nevertheless, nanopore sequencing has the potential to become a routine

analytical tool in the future.

17

In situ nucleic acid detection

In vitro methods can accurately quantify the simultaneous presence or absence

of different target sequences, but lack spatial information. Bulk measurements

cannot give information about gene expression patterns in heterogeneous tissues

as they mask the information by yielding an average expression profile. In situ

methods, however, allow the analysis of spatially-resolved gene expression

patterns in heterogeneous tissue. Such level of resolution is needed for example

in the study of the transcriptome in cancer tissue to direct treatment, as bulk

measurements can mask the existence of a minor population such as cancer stem

cells. Other application areas include the study of naturally heterogeneous

tissues such as brain tissue. In the following sections I will give a brief overview

of different technologies used to achieve spatially-resolved transcriptome

analysis. Common to most in situ techniques are the following steps: tissue

fixation and permeabilization followed by hybridization of probes. Signal

detection and/or amplification vary between the techniques described.

Fluorescent in situ hybridization

With the development of in situ hybridization (ISH) in 1969 it was possible to

study nucleic acids directly in cells. Gall and Pardue demonstrated the detection

of ribosomal DNA by hybridization of radiolabeled RNA67

. Advancements in

fluorescence microscopy have replaced radiolabeled probes with fluorescently-

labeled probes. In the original fluorescent in situ hybridization (FISH) protocol,

a fluorochrome is covalently bound to the RNA probe allowing DNA detection

by hybridization and subsequent fluorescent detection68

. Although the first FISH

protocols were less sensitive than autoradiography it had significant advantages:

increased spatial resolution and increased speed, making results available in one

day68

. mRNA detection, using biotinylated nucleotides in the probe sequence

and either antibodies or fluorescently-labeled avidin for detection, was

demonstrated by Singer et al. in 198269

.

Despite the potential of increased spatial resolution of FISH, compared to ISH,

which used radiolabeled probes, early protocols allowed only qualitative

information and not absolute counting of target sequences68

. The low signal-to-

noise ratio of long probes, partly caused by quenching of adjacent dyes, which

are randomly distributed along the probes, does not allow absolute

quantification70

. Improvements in probe design and imaging technology made

absolute quantitation with high spatial resolution possible. Instead of long,

randomly labeled probes, Femino et al. used several, adjacent to each other, 50-

base pair long probes that were labeled at predefined positions with five

fluorophores per probe and a GC content of ~50%. These improvements permit

18

counting of diffraction-limited spots, each arising from one single transcript71

.

Multiply labeled probes have been used for analysis of transcript distribution in

yeast64, 65

and mammalian cells72

, and for detection of transcription sites in

tissue73

. Differences in spot intensity due to incomplete hybridization or due to

only partially fluorescent probes impede the differentiation between true signals

and nonspecifically bound probes. Further optimizations, yielding higher

specificity and sensitivity, included construction of a probe library whereby each

probe is 17-22 nucleotides long and labeled with a single fluorophore74

. This

approach has been applied, amongst others, to simultaneously detect multiple

transcripts in mammalian single cells74

, for detection of fusion transcripts75

and

for the detection of stem-cell markers in tissue76

. Inclusion of modified

nucleotides, such as peptide nucleic acids (PNA) and locked nucleic acids

(LNA) increases probe binding stability and thus, probe lengths can be

shortened77, 78

. Using these modifications, short transcripts such as micoRNA

molecules can be specifically detected, although signal amplification is

necessary79, 80

.

The multiplexibility of single-molecule FISH (smFISH) is limited by the

number of fluorophores that can be spectrally distinguished. These limitations

have been bypassed by either using combinatorial fluorescent barcodes or by

sequential hybridization81, 82

.

Methods for signal amplification in situ

Traditional smFISH, due to low signal strength, requires high-magnification

objectives resulting in long acquisition times. The acquired signal can be

improved either by target or by signal amplification. In situ PCR and reverse-

transcription PCR (RT-PCR) have been demonstrated for target amplification,

but due to diffusion of amplification products this method is less quantitative83,

84. For signal amplification, either the signal, as in branched DNA (bDNA) or

the probe sequence, as in in situ RCA, can be amplified to obtain higher

sensitivity.

As in FISH, signal detection using bDNA relies solely on hybridization. Instead

of directly detecting mRNA molecules by hybridization of short fluorescently-

labeled probes to the target sequence a gene-specific probe, to which a pre-

amplifier binds, is hybridized85

. Multiple amplifier probes hybridize to the pre-

amplifier probe onto which labeled detection probes hybridize85

. The above

described mechanism of bDNA results in a local increase in fluorescence

without the need of target amplification. bDNA has been used for in situ

detection of viral DNA and mRNA in various cell types and in tissue85, 86

.

Another approach to obtain signal amplification combines immunoRCA with

ISH. A labeled probe is hybridized to its target sequence and detected by

19

immunohistochemistry combined with RCA87

. The bound antibody carries a

primer, which upon complementary binding of circular DNA initiates RCA and

amplifies the signal which is detectable by hybridization of fluorescent probes87

.

RCA is an isothermal amplification mechanism and the target molecules are

tethered to their amplification product, thus yielding bright diffraction limited

spots. Therefore, it is an ideal signal amplification method for in situ analyses,

which require high spatial resolution.

The specificity of in situ detection can be improved if detection of target

molecules does not depend solely on hybridization, but also on enzymatic

ligation. Padlock probes, amplified by RCA, have been used to detect DNA as

well as mRNA in situ41, 88, 89

. Larsson et al. demonstrated the detection of mRNA

molecules by first reverse transcribing the mRNA to cDNA. mRNA is then

digested by RNase H followed by hybridization and ligation of the padlock

probe to the cDNA target sequence (Figure 3)89

. Examples of application areas

include the differentiation of gene isoforms, detection of somatic mutations in

tissue and detection of viral RNA89-91

.

Figure 3. In situ rolling circle amplification. A) cDNA is synthesized from mRNA (in black) using

LNA modified primers (in grey). B) The mRNA is degraded by RNaseH and (C) a complementary

padlock probe hybridizes to its target sequence on the cDNA. D) The nick is sealed by a highly

specific ligase (in pink). E) Ligated padlock probes are locally amplified by a highly processive DNA

polymerase (in yellow). Fluorescently labeled oligonucleotides (orange stars) are hybridized to the

rolling circle product (RCP) and (F) visualized in a fluorescence microscope (RCPs in orange, nuclei

in blue).

20

In situ sequencing

Sequencing of the amplification products can be used to overcome the limitation

of the low multiplexity of traditional FISH, which is defined by the fluorophores

that can be spectrally resolved. In 2013, Ke et al. demonstrated targeted in situ

sequencing of RCA products allowing multiplex detection of mRNA transcripts.

Either an integrated barcode in the backbone of the padlock probe or a short

target site, filled by polymerization to circularize the padlock probe, can be

sequenced92

. Drmanac et al. applied the sequencing-by-ligation technology to

sequence RCA products generated in vitro93

. In situ sequencing is based on the

sample principle. An anchor primer is hybridized to the RCA products and a

sequencing library, consisting of random hexamers with one fixed position, is

ligated to it92

. Each base is encoded by one fluorophore. The simultaneous

sequencing of 31 probes, partly based on the commercially available

OncotypeDx Breast Cancer assay, has been shown in breast cancer tissue

sections92

.

A similar, but untargeted, approach has been described by Lee et al. Random

primers containing a sequencing adaptor are used for cDNA synthesis. After

RNA digestion and circularization of single-stranded cDNA, primers,

complementary to the sequencing adaptor, are used to prime the RCA. The RCA

products are covalently linked to cellular proteins and sequenced using the

SOLiD chemistry achieving a read length of 27 nucleotides. Partition

sequencing, using pre-extended anchor primers with mismatches at the ligation

site, is used to decrease signal density in order to discern single amplification

products. The authors demonstrated the simultaneous sequencing of thousands

of genes and their cellular localization94

.

21

Diagnostic methods used in

bacteriology and virology

Numerous methods are available that are commonly used for the diagnosis of

diseases of viral or bacterial origin. To give an overview of this broad field some

of the most widely used methods are described below. Requirements on methods

used in clinical practice differ regarding automation, sample handling and time

from methods used solely in a research setting. Cost-benefit analysis is

important in decision-making whether or not new methods should be

implemented into routine diagnostics. For a new method to be used for clinical

diagnosis it must not only be more sensitive or more multiplexed than currently

used methods, but time saving, labor and costs are equally important. Thus,

some methods have been used in routine diagnostics with little change for more

than 100 years albeit more sensitive but at the same time more expensive

methods exist.

Bacterial identification by culture and biochemical

methods

For many bacterial infections culture, either on solid or in liquid medium, is

considered the gold standard in microbiology with it beginning already in the

19th century. Robert Koch demonstrated in 1881 the use of solid media for

bacterial culture, which can further be used for the isolation of pure cultures95

.

This laid the basis for medical diagnosis of bacterial infections as pure cultures

are essential for correct pathogen identification and determination of antibiotic

susceptibility. Most bacterial culturable pathogens form colonies on solid media

in 24-48 hours, but slow growing bacteria such as Mycobacterium tuberculosis

require longer culture time due to long generation time. Escherichia coli, the

most common pathogen in urinary tract infections (UTI)96

, for example, has a

generation time of about 20 min97

whereas M. tuberculosis has a generation time

between 18 and 24 hours98

. Bacterial growth on agar plates allows phenotypic

identification by morphology and odor. A single bacterium can give rise to a

colony consisting of thousands of clones99

and thus, microbial viability can be

examined. The plate count method, counting the number of colonies grown on

an agar plate, allows quantitative analysis of a sample. With its simplicity and

high sensitivity of 10 bacteria per ml100

it is highly suitable for clinical

diagnostics. Despite protocol optimizations it is estimated that less than 2% of

all bacteria can be grown in culture99

. Non-culturable bacteria, such as the

syphilis causing bacterium Treponema pallidum, cannot be detected by culture

22

but must be detected by other means such as microscopic examination or by

molecular methods101

.

To further identify or confirm identification by morphology of a single culture,

several biochemical tests can be performed. A number of commercially

available systems for bacterial identification exist where biochemical tests are

incorporated into a strip format allowing simple inoculation and identification of

clinically relevant pathogens with a specificity of around 90%102

. Besides

limited specificity, the downside of these tests is the long turnaround time as

pure cultures are needed and the test itself requires an overnight incubation. In

recent years, automated systems such as the Vitek2 (BioMérieux) and the

Phoenix system (Becton Dickinson) have been widely used in large clinical

laboratories and replaced the above mentioned strip-based tests as they allow

automation and highly multiplexed analysis in a short period of time. For

optimal specificity and sensitivity a pre-culture is still needed103

.

Cultures combined with biochemical tests are commonly used in clinical

microbiology laboratories due to the high sensitivity, specificity and ease of use,

but the long turnaround time impedes fast diagnosis. The below described

methods are more rapid and have partly replaced identification solely based on

culture and biochemical tests, but usually have other drawbacks such as lower

sensitivity or higher costs.

Microscopy for rapid microbial diagnosis

Light microscopy combined with different staining methods can be used for

bacterial identification. Smear microscopy for diagnosis of tuberculosis (TB)

was developed more than 100 years ago104

allowing diagnosis of the most

infectious cases (>5,000 – 10,000 bacilli per ml sputum)105

. Due to its

simplicity, low demand on equipment and low cost per sample it is still the most

commonly used method, particularly in low resource settings. However, the

biggest drawback of smear microscopy is the low sensitivity, which is 50% of

culture106

, and the inability to establish drug resistance profiles.

Electron microscopy (EM) has been widely used for diagnosis in virology107

.

The high resolution of EM, due to the short wavelength of electrons, enables

imaging of viral particles108

. Only a simple negative staining, to increase

contrast between specimen and background, is required before visualization

using EM109

. As the staining procedure does not involve any pathogen specific

reagents, EM allows viral classification up to the family level without prior

knowledge of the infectious agent. A wide range of samples can be used for EM:

vesicle fluids, body excretions and biopsies. Sensitivity levels for EM are

around 105 particles per ml without the use of enrichment techniques

107 and time

from sample to readout can be as little as 15 min107

. Despite its rapidity and low

23

cost per sample EM requires highly skilled personal and highly expensive

equipment and thus, is not suitable for screening purposes. The emergence of

nucleic acid-based methods has greatly replaced EM in viral diagnostics, but it

is still partly used for urgent diagnosis.

Immunoassays

Like microscopy, immunoassays have been widely replaced by nucleic acid

detection methods for clinical diagnosis, but are still used in certain instances.

Since the development of the enzyme-linked immunosorbent assay (ELISA)110

and the enzyme immunoassay (EIA)111

in 1971 numerous assays based on these

principles have been used in clinical diagnostics. Briefly, ELISA is based on the

detection of an antigen by an enzyme-linked antibody. Upon binding and

subsequent washes to remove unbound antibodies, a chemical substrate for

detection is added110

. Application areas include the detection of the human

immunodeficiency virus (HIV)112

and detection of bacteria belonging to the

genus Borrelia, causing Lyme disease113

. Sensitivity and specificity can be

extremely high with >98% reported for some commercial HIV assays114

, but

might be insufficient for other applications115

.

Latex agglutination assay is another type of immunoassay that is used for

detection of pathogens, e.g., for rotavirus A detection116

. Latex beads are

sensitized with antigens forming visible aggregates upon antibody binding117

.

The same principle can be used to detect antigens by immobilizing the

corresponding antibody onto the beads. These assays are rapid and simple

without requiring expensive equipment or extensive training, but their

specificity and sensitivity depend heavily on the antibodies used. Furthermore,

only conserved epitopes can be targeted as antigenic drift can cause false-

negative results.

The drawbacks of immunoassays, such as low sensitivity, are partially addressed

by nucleic acid amplification-based testing methods, which have entered into

routine diagnostic settings.

PCR-based methods for pathogen identification

Quantitative PCR is increasingly being used for routine diagnostics of infectious

diseases as it offers several advantages over more traditional methods: fast

turnaround time, high specificity and high sensitivity. Microbial pathogens can

be directly detected in clinical specimens rendering culture unnecessary. In

bacteriology, diagnostic applications include panbacterial PCR118

, species

specific PCR119, 120

and antibiotic susceptibility testing (AST)121-123

. Although

PCR-based methods are used for detection of fast growing bacteria, culture

24

remains the reference diagnostic method in many cases. Quantitative PCR is

particularly suited for the diagnosis of slow growing or difficult to culture

bacteria, such as Mycobacteria120

or Chlamydia trachomatis119, 124

, and for life-

threatening conditions where rapid and correct treatment is essential as it

significantly reduces morbidity125

.

In contrast to bacteriology, PCR-based methods are widely used in virology as it

offers several advantages over traditional methods like virus isolation in cell

lines126, 127

. Cell culture for viral detection, which was regarded as the gold

standard for several decades, is slow and requires extensive technical expertise.

The introduction of qPCR to routine virology omitted the need of culture in

most instances offering rapid and sensitive diagnosis126

. Although considered as

the “new” gold standard there is a risk that PCR-based methods will fail to

detect highly variable viruses. As reported by Ripa et al., genetic drift can cause

failure in primer hybridization and thus, unsuccessful amplification128

.

Therefore, constant surveillance of circulating strains is necessary.

Mass spectrometry

Mass-spectrometry (MS) plays a growing role in clinical microbiology

laboratories as it is a powerful tool for identification of microbial biomarkers129

.

MS measures the mass in relation to charge (m/z). The substrate to be analyzed

is first ionized in vacuum and then separated in a mass analyzer containing an

electro-magnetic field. Separation is based on the m/z ratio of the ionized

particles and the ratio output is measured by a detector. A widely used mass-

spectrometer in clinical microbiology laboratories is the time-of-flight (TOF)

mass-spectrometer, which offers sensitive, parallel ion detection with a very

high mass range. The flight time of each particle to the detector is recorded

whereby heavier particles have a longer travel time130, 131

. Different ionization

techniques have been developed to enable the study of biological samples. One

commonly used technique is matrix-assisted laser desorption/ionization

(MALDI). In MALDI, the sample is first mixed with an appropriate matrix

followed by short laser pulses to desorb and ionize the sample for analysis in a

mass-spectrometer132

. The matrix thereby absorbs the radiation and prevents

fragmentation of large molecules133

. Thus, MS coupled to MALDI allows

analysis of complex biological matrices, as there is little or no fragmentation of

biomolecules during the ionization stage.

MALDI-TOF MS has entered clinical microbiology laboratories allowing

simple, rapid, and high-throughput analysis of patient samples for the presence

of pathogens. The spectrograms obtained are compared to a database for

pathogen identification. Already in 1975, Anhalt and Fenselau demonstrated the

use of MS for identification of bacteria134

. Since then there have been a number

25

of reports on the use of MALDI-TOF for reliable identification of bacterial

species135-137

but all of these studies required bacterial isolates. Seng et al.

observed an accuracy of 95.4% compared to conventional phenotypic

identification establishing its usability in routine diagnostics135

. In order to have

an even greater impact on clinical diagnosis, identification of pathogens directly

from body fluids is desirable. Direct identification has been shown on blood

culture samples but several washing and centrifugation steps were required138-

140. Overall, the specificity in these studies ranged between 75.8 and 80.4% and

required culture before analysis138-140

. Thus, further development such as

database updates and optimization of sample preparation protocols are needed

for accurate analysis of body fluids. As sensitivity of MALDI-TOF MS is low,

only body fluids containing a high bacterial load e.g., urine samples or blood

cultures can be analyzed.

Methods for antibiotic susceptibility testing

Antibiotic susceptibility testing is an important part of clinical microbiological

diagnosis as effective antibiotics determine treatment success. The most

commonly used methods rely on phenotypic analysis such as disk based

diffusion assays and microdilution assays. PCR is commonly used for genotypic

testing in research settings, but is not widely implemented in diagnostic

laboratories with an exception being drug susceptibility testing for TB.

The classic approach to AST is based on the determination of the minimum

inhibitory concentration (MIC), which is the lowest concentration of an

antibiotic that inhibits growth. In 1971, an international collaborative study on

antibiotic sensitivity testing tried to standardize testing procedures for

determination of MICs and recommended agar dilution and disk diffusion

assays141

, which are still commonly used in routine clinical microbiology

laboratories. The agar dilution method uses a series of agar plates with different

concentrations of antibiotics to determine the MIC for visible microbial

growth142

. A similar method, called broth dilution method, uses liquid medium

instead of agar plates. The disk based diffusion assay has the advantage that it is

technically quite simple and easy to perform by low-skilled users. Furthermore,

several antimicrobial substances can be tested on the same plate. Antibiotic-

impregnated disks are placed on an agar plate, inoculated with the strain to be

tested and incubated overnight. Zones of no growth are measured and zone

diameters are compared to published values to classify the tested strain as

susceptible or resistant143

. The agar dilution method is considered to be the

standard method and therefore, other methods need to be correlated to it142

. The

above described methods for AST require a pure culture as a starting point plus

at least one overnight culture. There is an obvious need for more rapid methods

26

that can readily be applied in the clinics. I will describe some of the most

promising and widely used technologies below.

Instead of phenotypic determination of antibiotic resistances, qPCR can be used

for genotypic analysis detecting the presence of resistance genes or point

mutations causing resistance. There have been several reports in the last two

decades describing the use of qPCR for AST144-147

. One widely used qPCR

system for drug resistance detection in TB is the GeneXpert MTB/RIF test

(Cepheid), endorsed by the World Health Organization (WHO) in 2010148

. This

system has the advantage of a fully integrated sample processing from nucleic

acid isolation to amplification and detection149

and thus limiting cross-

contamination and hands-on time. Furthermore it has a significantly improved

turnaround time of two hours120

compared to conventional culture based testing,

which can take weeks to months in the case of M. tuberculosis98

. Although much

faster than the methods described above, the major disadvantage of PCR-based

tests is that novel, previously unknown resistance genes will be missed as they

will not be covered by the PCR primers. In addition, the presence of a resistance

gene does not automatically imply phenotypical resistance150

. Furthermore,

multiplexing and thus, targeting all relevant mutations requires extensive

optimizations.

Recently, MALDI-TOF MS has been used to phenotypically detect resistances

towards β-lactams151-155

. Detection by MS for these classes of antibiotics is

possible by monitoring the mass peak for the antibiotic and their corresponding

hydrolyzed product154

. Absence of the peak of the hydrolyzed product

corresponds to susceptible bacteria, as they do not synthesize the enzymes able

to degrade β-lactams. The main limitation of this technique is the requirement of

a fresh culture, usually grown overnight156

. In order to be considered a general

method for AST, the detection of resistance mechanisms where the antibiotic

uptake is reduced or the drug is pumped out of the cell and the detection of

multi-resistant bacteria must be further developed as current methods are either

too labor-intensive and expensive or are unable to accurately quantify antibiotic

concentrations156

.

Several methods for ASTs have been developed using microfluidic

technologies157-159

. Cira et al. and Choi et al. used a microfluidic device that

visually monitors the growth of bacteria in the presence of antibiotics158, 159

.

Microfluidic approaches offer the advantage that several antibiotics and

concentrations can be tested in parallel with a very low number of bacteria

required but they most often lack species identification and thus additional

methods are needed.

27

Summary of diagnostic methods

Below is a brief summary of the above described methods (Table 1). As can

be seen in the table each method has its advantages and disadvantages and

thus, must be carefully chosen for each diagnostic question and depending

on available resources.

Table 1. Comparison of widely used diagnostic methods in bacteriology and virology

Method Time

(approx.)

Pros Cons Refs

Culture (bacteria)* Overnight Sensitivity (10

bacteria per ml)

AST is possible

Trained personnel for

identification required

Only 2% of all bacteria are

culturable

99, 100

Light microscopy

(bacteria)

Few hours Applicable to low

resource settings

Short time

Sensitivity (5,000-10,000

bacilli/ml for M. tuberculosis)

No AST possible

105

Electron

microscopy (virus)

15 min-1

hour

Short time

No prior knowledge

of virus needed

Sensitivity (105 particles/ml)

Low throughput

Skill-based

High equipment cost

107

Immunoassay

(virus)

30 min-1

hour

Short time

Simplicity

Suitable for

automation

Specificity depends on available

antibodies

Antigenic drift can cause false-

negatives

Low multiplexity

160

qPCR (virus) 2-3 hours Sensitivity

Specificity

High-throughput

Suitable for

automation

Antigenic drift can cause false-

negatives

Low multiplexity

Trained personal required

Cost per sample

128,

160

MALDI-TOF MS

(bacteria)

10 min Short time

Accuracy

Simplicity

High-throughput

No prior knowledge

of bacteria needed

Sensitivity (subculture of 105

bacteria is required)

High equipment costs

Certain bacterial species are

difficult to distinguish

135,

161,

162

*The pathogenic agent in brackets denotes the pathogen that the method is mainly used for

28

Methods for prenatal diagnosis of

chromosomal aneuploidies

Trisomies are the most common chromosomal anomalies in humans, mainly

caused by non-disjunction during meiosis. Trisomy 21, known as Down

syndrome, is the most common one163

, occurring in 1 in 800 live births164

,

making up 30-40% of all chromosomal abnormalities163

. The second and third

most prevalent trisomies are trisomy 18 (Edwards syndrome), followed by

trisomy 13 (Patau syndrome). As chromosomal aneuploidies have severe effects

on the offsprings’ health, several prenatal diagnostic and screening methods

have been developed to aid in prenatal counseling.

Molecular, non-invasive, diagnostics offer several advantages compared to more

traditional, invasive techniques. The biggest advantage of accurate non-invasive

techniques is to render invasive procedures unnecessary, which are associated

with a certain risk for procedure-related pregnancy loss165, 166

. In addition to the

information gained regarding chromosomal aberrations, targeting nucleic acids

with single nucleotide resolution can yield information about other potential

health defects, e.g., mutations causing cystic fibrosis.

Traditional prenatal testing

Traditionally, prenatal screening methods rely on the measurement of several

biochemical markers in maternal blood and on ultrasonography. For definite

diagnosis invasive procedures are needed. Already, in 1984 an association

between low maternal serum alpha-fetoprotein (MSAFP) levels and

chromosomal abnormalities was reported167

. An increase in nuchal translucency

was first correlated with chromosomal defects in 1992168

. Several options for

prenatal screening for fetal chromosomal abnormalities to risk-stratify patients

are available, e.g., measurement of nuchal translucency thickness and

concentration of maternal serum biochemical markers such as the pregnancy-

associated plasma protein-A (PAPP-A), maternal serum free β-human chorionic

gonadotropin (β-hCG), inhibin A, unconjugated estriol and MSAFP169

. Upon

presentation for prenatal screening in the first trimester combined first and

second trimester screening should be offered as it has the highest detection rate

of chromosomal aneuploidies170

. The quadruple test alone, consisting of protein

concentration determination of MSAFP, hCG, unconjugated estriol and inhibin

A has a detection rate for Down syndrome of about 80%170

. By combining this

first trimester screening with measurement of nuchal translucency and

measurement of PAPP-A detection rates for Down syndrome of up to 96% can

be achieved170

.

29

Upon receiving a high risk score genetic counseling in combination with a first

trimester chorionic villus sampling (CVS) (at about 9-11 weeks of gestation) or

a second trimester amniocentesis (at about 15 weeks of gestation) should be

offered169

. Traditionally, chromosome diagnosis was done by karyotyping.

Condensed chromosomes, present in the metaphase, are stained by Giemsa

banding and analyzed in a microscope171

. Since amniotic fluid does not contain

dividing cells a cell culture must be performed first and thus, delays diagnosis.

Cell culture is also used to confirm diagnosis on cells obtained directly by

CVS172

. Molecular methods for prenatal diagnostics such as FISH and

quantitative fluorescent PCR (QF-PCR) provide a faster turnaround time.

Although faster than karyotyping, FISH is labor intensive and requires intact

cells to accurately detect chromosomal aberrations173

. QF-PCR on the other

hand requires the presence of polymorphisms such as short tandem repeats and

is less informative than karyograms172

. Despite newly developed techniques

chromosome karyotyping via Giemsa banding remains the gold standard for

detection of aneuploidies from invasive samples with a false negative rate of

below 1%174

. Although conventional chromosome analysis has a very high

detection rate, small but significant risks are associated with the necessary

invasive procedures. Procedure-related pregnancy loss rates after second-

trimester amniocentesis has been estimated to be about 1 in 370-600165, 166

and

similar loss rates are estimated for CVS166

. Furthermore, the long turnaround

time of traditional techniques render non-invasive prenatal testing (NIPT), an

attractive alternative. In addition, waiting times for screening results between the

first trimester and second trimester screening can cause psychological burden on

the patient.

Non-invasive prenatal testing

Fetal cells have been recovered from maternal plasma for the first time already

in 1969175

and the feasibility of using these to detect Down’s syndrome has been

demonstrated176, 177

. Nonetheless, they have not been extensively used in

prenatal diagnostics due to their rarity, limiting robust detection178

. In 1997, Lo

et al. discovered cell-free fetal DNA (cffDNA) in maternal plasma and serum179

.

The presence of large amounts of background maternal DNA has posed

challenges to the development of accurate NIPD technologies. It has been shown

that cffDNA concentration correlates with gestational age180

. The small fraction

of fetal DNA, up to 10 % in gestational week 10181

, in maternal blood makes it

challenging to detect chromosomal aneuploidies using cffDNA as its sequence

content is nearly identical to maternal DNA. Evidence exists that the majority of

cffDNA is derived from apoptotic cells as it is enriched for nucleosome-bound

fragments182

with a size peak at <200-300 bp, shorter than cell-free maternal

DNA182-184

. cffDNA has a relatively short half-life of less than two hours and is

cleared rapidly from circulation after delivery174, 175

. Hence, it is well suited for

30

prenatal diagnosis as no fetal DNA from former pregnancies will be present at

the point of testing. Fetal progenitor cells, on the other hand, have been detected

in maternal blood even 27 years postpartum185

. Several different non-invasive

methods using cffDNA have been developed in the last couple of years with the

majority being sequencing-based. Below I describe approaches that are based on

differential methylation to distinguish fetal from maternal DNA, approaches that

use massively parallel sequencing (MPS), and approaches using digital PCR. I

will then give a short overview of commercially available assays for NIPD

detecting chromosomal aberrations.

The methylation pattern between placental-derived and maternal-derived DNA

differs and several markers have been discovered that can potentially be used in

NIPD186, 187

. Several methods have been reported using methylation markers for

NIPD of trisomies188-190

. Approaches used include bisulfite conversion and

sequencing of PDE9A, a gene on chromosome 21 that is completely methylated

in blood but unmethylated in placenta188

, fetal DNA enrichment using

methylation-specific antibodies combined with qPCR189

and bisulfite conversion

combined with methylation specific PCR190

. These studies based on differential

methylation patterns had significant drawbacks such as low sensitivity188, 190

,

high false-positive rates188, 190

or showed low reproducibility191

leading to poor

diagnostic performance.

Fan et al. and Chiu et al. demonstrated the use of MPS to precisely enumerate

DNA fragments in a locus-independent fashion and thereby detecting fetal

aneuploidies in maternal blood without any further enrichment of fetal DNA182,

192. The large number of counted sequences allowed robust diagnosis of fetal

trisomy 21, detected by the overrepresentation of the corresponding sequence

tags182, 192

. Chromosomes with a high GC content showed a large variation in

sequence counts and thus, might have limited detection sensitivity. Chromosome

13, 18 and 21 showed low variance making them suitable for NIPD using DNA

sequencing182

.

Digital PCR on the other hand is technically challenging as it requires thousands

of PCR reactions to correctly detect trisomy 21 in samples having low fractional

DNA concentrations193, 194

. Thus, for accurate diagnosis additional enrichment of

fetal DNA sequences from maternal plasma is required.

In the last couple of years several tests for NIPD have been commercialized but

have not yet been implemented into routine diagnostics. Most commercially

available tests for NIPD rely on next-generation sequencing as sequencing-

based technologies proved to be advantageous in terms of sensitivity, precision

and robustness195

. The US-based companies, Verinata and Sequenom, use non-

targeted MPS technologies whereas Natera uses targeted SNP-based sequencing

and until recently Ariosa’s technology was based on chromosome selective

31

sequencing196-199

. In 2014, Ariosa demonstrated that their chromosome targeted

approach in combination with microarrays yields more accurate results than

when combined with sequencing technologies200

. Costs for a single test range

approximately between $800 and $2,700199

contributing to the fact that NIPT is

not yet implemented for population-wide prenatal screening.

32

Molecular Diagnostics in Oncology

Cancer is a disease characterized by a large degree of heterogeneity, exhibiting

intra- as well as inter-tumor heterogeneity201-203

. Underlying causes of the

observed heterogeneity include numerous somatic mutations, such as point

mutations, copy number variations and chromosomal rearrangements202, 204, 205

.

These molecular signatures characterize a cancer and can offer valuable

information about the best treatment course. However, detailed characterization

of these signatures is not possible by the widely used TNM staging system,

which stands for tumor, node and metastasis. The tumor is examined based on

size, the proximity to lymph nodes and the presence of metastasis. Molecular

methods, on the other hand, can give information about the mutational landscape

of a tumor which can be used to estimate the likelihood of recurrence in breast

cancer patients206

or response to therapy206

. Additionally, information on the

origin of the tumor207

can be gained from these signatures. Evidence exists that

target-based treatment strategies are only effective in a subpopulation of cancer

patients and thus, knowledge of the mutational status of a tumor is essential for

effective targeted therapy208, 209

. Predictive biomarkers include mutations in

cancer driver genes, such as in the epidermal growth factor receptor (EGFR) in

lung cancer, changes in methylation, and changes in expression levels as

observed for the estrogen receptor in breast cancer208

.

About a decade ago a number of molecular tests for gene expression profiling in

cancer emerged on the market. Many of these tests for cancer diagnostics are

based on PCR or microarrays. Sequencing-based technologies are often only

used as a discovery tool or for confirmatory purposes, but are expected to play

an increasing role in cancer diagnostics in the near future. Perou et al.

demonstrated a large heterogeneity in expression pattern in breast cancer210

.

Based on the observed heterogeneity several assays have been developed for

risk stratification of patients206, 211-214

. Genomic Health’s Oncotype Dx, a

multigene breast cancer assay, has been incorporated into clinical guidelines of

the American Society of Oncology215

. The Oncotype Dx assay determines the

risk for recurrence and response to therapy in breast cancer by examining 21

genes in a RT-PCR-based assay206

.

Most molecular tests used in the clinical diagnosis of cancer do not have single

cell resolution as nucleic acids are analyzed in bulk measurements. Thus, spatial

information and detailed information on heterogeneity are lost. Sanger

sequencing has been used for mutation detection in the Kirsten rat sarcoma viral

oncogene homolog (KRAS). Although, in samples containing less than 10%

tumor cells a significantly lower mutation rate was reported and thus, requiring

manual microdissection for tumor enrichment216

. Newly developed assays,

33

mostly PCR-based, have reported improved sensitivities of as low as 1% mutant

alleles217

. But nonetheless, these molecular methods must often be accompanied

by histopathology to guarantee accurate diagnosis. In situ techniques are able to

combine molecular testing and histopathology into one test and thus, could

eliminate the requirement of several assays.

One example of in situ techniques for cancer diagnosis is the use of FISH for

gene expression analysis in breast cancer patients, as different patterns are

associated with different clinical outcomes218

. Overexpression of the human

epidermal growth factor receptor 2 (HER2) is commonly measured since it is

positively correlated with response to trastuzumab, a monoclonal antibody

binding to HER2 and thus inhibiting proliferation219

. As described earlier, FISH

is an excellent method for low-plex gene expression analysis in situ. However, it

does not offer single nucleotide resolution needed for the analysis of point

mutations that might significantly affect treatment success.

Padlock probes, on the other hand, are a reliable tool for detection of point

mutations in situ. The potential of padlock probes for clinical application has

been demonstrated by mutational analysis of the most common sample

preparations used in diagnostic routine: fresh frozen tissue, formalin-fixed,

paraffin-embedded tissue, tissue microarrays and tumor touch imprints. A

mutation detection rate of 1% makes the padlock probe technology a potential

tool for clinical diagnosis of tumor samples90

.

34

Present investigations

Detection of rotavirus using padlock probes and rolling

circle amplification

In this work, we applied padlock probes to detect a highly variable double

stranded RNA virus in clinical samples.

Rotavirus is a highly contagious virus, causing diarrhea. By the age of five

nearly every child has experienced at least one episode of rotavirus infection220

.

Rotavirus is the number one pathogen recorded for hospitalizations due to

gastrointestinal diseases221

and contributes to a high number of deaths, mainly in

developing countries222

. Thus, the financial burden of rotavirus infections is high

and simple and accurate diagnostic methods are needed to control its outbreak,

and for surveillance purposes. Diagnosis is usually achieved by a latex

agglutination assay. Although the latex agglutination assay is rapid and easy to

perform, it lacks sensitivity223

. RT-PCR, which is mainly used in research

settings, overcomes this limitation, but due to the highly variable nature of

rotavirus, strain variants can be easily missed if not covered by the primers.

In this study we designed a pool of padlock probes covering ~95% of the

published rotavirus A sequences in the NCBI database. The pool consisted of six

padlock probe mixes whereby four of these included degenerated bases creating

a total number of 58 unique padlock probes. As padlock probes are easily

multiplexed and amplified independently of each other, new strain variants can

be included in the assay by a simple addition of new padlock probes to the

already existing pool. RNA was extracted from fecal samples and transcribed

into cDNA using degenerated primers able to cover a large part of rotavirus

sequences. RNA/cDNA hybrids were heat denatured and the cDNA was

subsequently captured on magnetic beads. Padlock probes were hybridized and

ligated at temperatures significantly higher than the melting temperature of each

probe arm to ensure specific ligation. Unbound probes were washed away as

they inhibit the subsequent amplification reactions. Ligated and circularized

padlock probes were amplified by RCA. To increase the amplification factor,

RCPs were monomerized by restriction enzyme digestion and a second round of

RCA was carried out. Fluorescently labeled RCPs were detected in a

microfluidic setup using an optical imaging system.

With the setup described, we achieved a sensitivity of 1,000 synthetic targets

equivalent to the cDNA. To evaluate the efficiency of a multiplexed assay,

different numbers of probes were added and amplified. A decrease in signal with

an increase in probe number was not observed. Thus, inhibition of competing

35

probes could be ruled out. Twenty-two clinical samples were tested with our

assay, including two rotavirus negative samples. Seventeen of these samples

were diagnosed as positive and confirmed by a PCR-based assay using agarose

gels as a readout format except for one sample that resulted positive only by the

padlock assay. The remaining three rotavirus positive samples were neither

detected by our assay nor did they yield a specific band on the gel. This might

be explained by failed cDNA synthesis due to low RNA quality or due to

mismatched primers.

To summarize, we demonstrated a highly variation tolerant assay for detection

of viral RNA using padlock probes and RCA. The whole assay from cDNA

synthesis to readout can be completed within three hours. Our assay has

advantages over RT-PCR as it can be highly multiplexed to cover a wide range

of strain variant without loss in efficiency.

A general method for rapid determination of antibiotic

susceptibility and species in bacterial infections

In this work, we established a rapid assay for simultaneous species identification

and AST directly in clinical samples.

Extensive use and over-prescription of antibiotics has led to an increase in

selective pressure on bacteria and contributed to the spread of antibiotic

resistances224

. Resistance mechanisms develop rapidly and no novel class of

antibiotics has been discovered in the last 25 years. The WHO recently

published a report underlining the alarming rate at which pathogens become

resistant and thus, impact our ability to treat common infectious diseases225

. In a

post-antibiotic era, previously curable diseases might turn to be deadly once

again. As traditional identification of bacterial species and determination of

antibiotic susceptibilities is slow, antibiotic treatment is often empirical and

thus, ineffective drugs might be prescribed. Therefore, rapid AST is essential to

maximize optimal use of antibiotics.

We merged the traditional culture based diagnostics with molecular methods for

rapid identification and quantitation to achieve AST and demonstrated its use for

diagnosis of UTI. We designed padlock probes for the common urinary

pathogens, E. coli, Pseudomonas aeruginosa and Proteus mirabilis, targeting

the 16S rRNA gene. Samples were split into several vials and cultured in the

presence and absence of antibiotics for a short period of time. Subsequently

bacteria were lysed by simple addition of sodium hydroxide and heat. The

targeted DNA sequences were captured on magnetic beads followed by a quick

wash to remove the sample matrix. The species specific padlock probes were

hybridized and ligated in order to be amplified by C2CA. For detection, an

36

optical imaging system was used allowing digital quantification of RCPs.

Comparison of counts of antibiotic containing samples with the sample lacking

antibiotics established the antibiotic resistance profile (ASP) of the targeted

bacterial species.

We first investigated the time necessary to detect bacterial growth. Using our

padlock probe-based method growth could already be detected after 30 min of

culture whereas a viable count did not detect any growth before 90 min of

culture. Second, we tested the specificity of the padlock probes by parallel

incubation with the three target bacterial strains. No unspecific signal was

observed for non-matching padlock probes. To establish an ASP for E. coli for

ciprofloxacin and trimethoprim, three different strains were tested. As expected,

resistant strains showed growth in the presence of the antibiotic they were

resistant to. Only minor growth was observed in the presence of the antibiotic

for which the tested bacterial strain lacked resistance mechanisms. A first set of

32 patient samples was used as a training set to establish cut-off values for

determination of antibiotic resistances in E. coli. The developed algorithm was

verified by a blind prospective study comprising 56 urinary samples. Species

identification for E. coli was correct in 55 out of the 56 analyzed samples with

the 56th

sample having fewer bacteria than required to be considered a UTI226

.

No false-positives were observed and resistance profiles were correctly

identified for all samples containing E. coli above the threshold for UTI.

Furthermore, all results were in concordance with the routine testing performed

at a clinical laboratory.

In conclusion, we have established an assay that is not limited to UTI, but can be

adapted for AST in other infections, such as TB or sepsis. The number of

antibiotics tested can be increased by simply adding an extra vial per antibiotic.

Additional pathogens can be targeted by either a parallel or multiplexed

approach using a multicolor detection system rendering this assay suitable for

general bacterial identification and AST.

Elimination of maternal DNA for accurate non-invasive

prenatal testing: a pilot study

In this work, we have established an assay for detection of trisomy 21 and 18

that uses differential methylation for enrichment of fetal DNA and padlock

probes amplified by RCA for precise quantitation.

Traditional prenatal diagnosis for chromosomal abnormalities rely on screening

methods, such as measurement of biochemical markers and nuchal

translucency169

. If high-risk scores are obtained, invasive methods can be used to

confirm diagnosis169

. Invasive methods are however associated with a certain

37

risk for procedure-related abortions165, 166

. The discovery of cff-DNA179

has

initiated intensive research to develop precise and reliable tests for NIPD with

the goal to render invasive tests unnecessary. In the last couple of years

numerous methods, mainly sequenced-based, have been developed195

. A

drawback of these methods are the laborious nature and high cost. We therefore

propose a padlock probe-based assay achieving precise enumeration of target

molecules by RCA and fluorescent detection.

Target sites, that are hypermethylated in placental-derived DNA and

hypomethylated in maternal DNA were chosen186, 187

. All sites included the

recognition sequence for HpaII, a methylation sensitive restriction enzyme.

Before probe hybridization and ligation, samples were incubated with HpaII

cleaving unmethylated maternal-derived DNA and thus, inhibiting probe

ligation and subsequent amplification. Ligated padlock probes were amplified

and detected using a confocal microscope or an automated, commercially

available two-color imaging system. A ratio of signals received from the two

chromosomes was calculated and used for determination of trisomy.

An excellent sensitivity of between 30 and 300 genomic equivalents was

achieved with the above described assay. We observed a decrease in variation

with an increase of counted objects, yielding a coefficient of variation of below

5% for 3,000 genomic equivalents. Female DNA, reflecting maternal DNA, was

used to determine the enrichment factor obtained by HpaII digestion. A three-

and five-fold enrichment for chromosome 21 and chromosome 18, respectively,

was obtained by using HpaII. Fourteen CVS samples, including four trisomy 21

samples and four samples with non-targeted chromosomal abnormalities, were

analyzed. The use of a cut-off value allowed correct identification of all trisomy

21 samples. When CVS samples were spiked into female DNA, a fetal fraction

of 30-40%, was required for accurate diagnosis.

To conclude, we have developed an assay that allows correct identification of

trisomy 21 and 18 in invasive samples based on precise quantification of RCPs.

With slight adaptations, such as increasing the number of differentially

methylated target sites, it should be possible to increase sensitivity and thus,

precision for robust and accurate NIPT.

Oligonucleotide gap-fill ligation for mutation detection

and sequencing in situ

In this work, we developed a novel approach for in situ mutation detection using

gap-fill ligation. Specificity, in this assay, relies on the requirement of two

ligation events to occur in order to circularize a padlock gap probe. Furthermore,

the generated RCPs can serve as a substrate for in situ sequencing.

38

The high specificity of ligases can be utilized to distinguish single nucleotide

differences45, 46

. Oligonucleotide gap-fill ligation combined with padlock gap

probes and RCA can be used to visualize SNVs or point mutations in situ.

Padlock gap probes are similar to conventional padlock probes, but differ at the

ligation site. Instead of directly ligating the two target complementary arms, a

short gap oligonucleotide is ligated to the padlock arms. Hence, two ligation

reactions instead of one are necessary to circularize the padlock gap probe. The

use of short gap oligonucleotides ensures specificity and offers a simple way to

distinguish between mutant and wild-type sequences.

We designed padlock gap probes, spanning a gap of six nucleotides, and a pool

of the corresponding gap oligonucleotides to target the mitochondrial A3243G

mutation causative of mitochondrial encephalomyopathy, lactic acidosis, and

stroke-like episodes syndrome (MELAS)227

and an expressed SNV in the ACTB

gene. Furthermore, we targeted a mutational hotspot in the EFGR gene

indicative for treatment response. Two requirements are necessary to obtain a

signal with our approach. The targeted mutation or SNV must be present and the

respective gap probe needs to be phosphorylated at the 5’ end in order to be

ligated. Thus, absence or presence of a signal was used to discriminate closely

related sequences. We evaluated the assay for efficiency and specificity using a

pool of gap probes. The specificity of the gap-fill ligation was further validated

by in situ sequencing.

A mutant cell line for the A3243G mutation and a wild-type cell line were tested

with the gap-fill approach. A total of seven gap probes were used and signal was

only detected in the wild-type cell line if the fully complementary gap probe was

phosphorylated. Phosphorylation of the mutant gap probe did not yield any

signal on the wild-type cell line, but on the cell line carrying the targeted

mutation. We estimated an efficiency of ~80% for the padlock gap probes

compared to conventional padlock probes. We further showed the applicability

of our approach to mRNA by specific detection of an SNV in mouse and human

fibroblasts. Addition of the gap probes, all being phosphorylated, generates

substrates for in situ sequencing making this assay suitable for simultaneous

multiplex mutation detection. We successfully genotyped the SNV in ACTB in

human and mouse fibroblast cells using gap-fill ligation combined with in situ

sequencing. Furthermore, we detected a clinically relevant point mutation in the

EGFR gene in lung cancer tissues. Specificity of the gap-fill ligation was

confirmed by in situ sequencing.

This assay design has the advantage of offering a simpler and cheaper design for

multiplexed mutation detection compared to the use of a pool of padlock probes

targeting several mutations in a short stretch of nucleotide sequence. The double

ligation ensures high specificity with minimal loss of efficiency. Potential

application areas include screening for mutations in mutational hotspot areas.

39

Concluding remarks and future

perspective

In this thesis, I have demonstrated the use of padlock probes combined with

RCA for a wide spectrum of diagnostic purposes. Although the novel methods

described in this thesis are not yet ready for implementation into routine

diagnostics, they certainly have the potential for application in clinical

diagnostics especially if they can be automated and thus, minimize manual

labor. Automation, labor time and cost are important factors one must consider

before employing new techniques in a clinical setting. Several efforts have been

undertaken, by our group and others, in order to automate padlock-based RCA

protocols, both in vitro and in situ228-231

. However, they still require further

development to allow sample processing in a completely automated fashion.

This can, for example, be done by implementing the whole protocol on a

pipetting robot or by designing a dedicated microfluidic chip for each assay.

Besides automation, additional parameters must be fulfilled until a laboratory

test can be offered for clinical diagnosis. In the US, laboratory-developed tests

that provide information on diagnosis and treatment options must adhere to the

Clinical Laboratory Improvement Amendments (CLIA)232

. Similar guidelines

exist in other countries. CLIA requires the verification and validation of the

accuracy and reliability of a diagnostic test to ensure the validity of clinical test

results232, 233

. Thus, the assay developments described in this thesis are still at the

very beginning on the potential road to a clinical diagnostic laboratory.

In summary, I have described the development of padlock probe-based assays

for use in molecular diagnostics in four different application areas. The

application areas, described in this thesis, are quite diverse, demonstrating the

wide applicability of padlock probes and rolling-circle amplification in the

diagnostic field. Depending on the diagnostic question padlock probes might be

an interesting alternative to already existing methods as they have advantageous

properties in regard to specificity and multiplexity, and additionally offer local

target amplification and digital quantification. If used in the right context,

padlock probes combined with RCA can generate powerful diagnostic tools

overcoming limitations of techniques currently employed in diagnostics.

40

Populärvetenskaplig

sammanfattning på svenska

Hur framgångsrik en medicinsk behandling blir beror ofta på tillgången till

exakta och tillförlitliga diagnostiska analyser för att vägleda läkare i val av

behandlingsmetoder. Ett optimalt test utmärker sig med avseende på specificitet

och känslighet. Beroende på användningsområdet kan snabbhet, låg kostnad och

enkelhet vara lika viktiga kriterier. För infektionsdiagnostik är analystiden

avgörande eftersom tid till påbörjad behandling oftast är kritisk för

tillfrisknande. Inom andra områden är tidsaspekten av mindre betydelse, till

exempel inom icke-invasiv fosterdiagnostik, en metod med vilken man testar för

fosterskador och ärftliga sjukdomar. Inom fosterdiagnostiken är specificitet och

känslighet de viktigaste parametrarna eftersom en feldiagnos kan få stora

konsekvenser.

I denna avhandling beskriver jag utvecklingen av fyra olika metoder avsedda för

molekylär diagnostik, alla baserade på så kallade ”padlock prober” (molekylära

hänglås) och rullande cirkel amplifiering. Trots att de utvecklade metoderna är

avsedda för olika tillämpningsområden, såsom diagnostik av infektiösa

sjukdomar, fosterdiagnostik och onkologi, har de gemensamt att de övervinner

vissa begränsningar som nu tillgängliga diagnostiska metoder har. Denna

avhandling innehåller två nya tester som riktar sig mot smittämnen: ett test för

att specifikt detektera rotavirus, ett mycket variabelt dubbelsträngat RNA-virus

som orsakar bland annat diarré. Det andra testet utgör ett nytt analysformat för

antibiotikaresistensbestämning vilket är snabbt och generellt tillämpbart för

olika patogener. Vidare beskriver jag utvecklingen av en metod för att anrika

fostrets DNA ur moderns blod, som använder DNA metylering som markör för

fostrets DNA. Detta DNA används för att exakt mäta kromosomförhållanden

och därmed upptäcka trisomi 21 och 18, vilket innebär en extra kopia av en viss

kromosom hos fostret. Den fjärde metoden som beskrivs i denna avhandling

använder speciella hänglåsprober för att upptäcka diagnostiskt relevanta

punktmutationer med hög specificitet direkt i celler och tumörvävnad.

De presenterade metoderna har potential att användas inom klinisk diagnostik,

efter automatisering av protokollen samt studier som validerar och verifierar

resultaten. Dessutom visar dessa metoder på den breda tillämpbarheten av

hänglåsprober som genom sina egenskaper avseende specificitet och

multiplexitet, är utmärkta verktyg för specifik detektion av nukleinsyra i provrör

och i celler.

41

Acknowledgments

This thesis would not have been possible without the contribution and support of

many others and I would like to express my sincere gratitude to everybody

involved in one way or another:

My supervisor Mats for accepting me as a PhD student and introducing me to

the world of padlock probes and RCA. Thank you for all your inspiration,

encouragement and endless optimism, never doubting that I will finish in time!

Thank you for believing in me! I really feel lucky I was able to pursue my PhD

studies in your group.

I spent the first two years of my PhD studies in Uppsala and would like to thank

Ulf Landegren, Ola, and Masood for creating such a nice and inspiring

research environment! It has been a great place to work in.

Dan Andersson, thank you for all your enthusiasm and for always making me

feel welcome in your lab! It was great getting the chance to work with you on

the UTI project!

I also would like to thank my collaborators who have contributed to the work

presented in this thesis: David, Christina and Jonas, for working together on

the rotavirus project. Erik, Anna, Eva, David and Jenny for the wonderful

collaboration on the UTI project. Erik, thanks for teaching me everything I

needed to know about microbiology, for coming in at the weekend to open the

door for me and sharing your lab space with me (sorry for the mess…)! Thank

you Eva for introducing me to routine clinical diagnostics and Jenny for always

having good advice on anything related to RCA. Maj, Lucia, and Stephanie,

for the valuable collaboration on the trisomy project. Thank you Maj for all

your input and comments. You are a truly inspiring scientist! Marco, Xiaoyan,

Chatarina, Linnea and Johan, thank you for the great collaboration on the gap-

fill project!

To the present and past members of the Nilsson lab for creating such a nice

place to work in and for all the fun in and outside the lab: Marco, thank you for

everything! For never getting tired of listening to my complaints, for always

trying to cheer me up, for the shopping trips, for the good advice I received

during the years and for everything else which I would neither have the time nor

the space to mention here! I don’t know how I would have survived without

you! ;-) Anna, thank you for being such a great friend and colleague! For all the

laughter, for sharing the highs and lows of life and work, for the glasses of wine

and for offering me samples for my diagnostic projects ! Iván, thanks for

being the number one resource in any chemistry-related question, for the best

42

popcorn of all times and for bringing me, together with all my stuff, safely

home! And of course, for being super enthusiastic about the boat! You will be a

great marinero! Annika, thanks for taking such good care of the lab, for the

great road trip and for always lending an ear for all and everyone! David, thank

you for introducing me to the lab and teaching me everything about C2CA and

for the fruitful collaborations! Malte and Elin L, thanks for the great

snowboarding trip to Järvsö! It was really fun! Malte, thanks for all the great

talks, for sharing lab bench and for being enthusiastic about our projects (at least

most of the time )! Elin L, for sharing a room at the conference, for the nice

conversations and for organizing the lab! Tom, thanks for the yummy pirogi, the

delicious hazelnut vodka and for taking good care of our very demanding

Aquila! Thomas, thanks for the nice chats, the recommendations on restaurants

and pubs and with Iván and Malte for supplying the lab with home-brewed

beer! Camilla, thanks for supervising with me my first student and all the help

on the magnetic projects! Xiaoyan, thanks for your help on the gap-fill project

and sorry for stressing you out about it! Thanks, Di for being such a nice desk

mate, for the interesting talks on science and keeping me updated in the field of

NIPT! Pavan, thanks for the great moving-in party and the nice Indian food!

Jessica, thanks for the nice chats at lunch and in the lab! Tagrid, thank you for

your encouragements and for always having a smile on your face! Rongqin,

thank you for sharing your knowledge! You always had an answer to any RCA

related question! Amel, thanks for the nice banana cupcakes and conversations

over lunch! Lotte and Elin F, thank you for the great time in Barcelona and for

bringing me to see Flamenco! Chenglin and Sibel it is great you have joined

this group. Thanks to Mustapha, Eva and all other students for contributing to

the nice atmosphere we have in the lab!

For the Uppsala people: Ola, Karin, Björn, Linda, Johan V, and Carl-

Magnus, thank you all for the great swimming sessions! Karin, thanks a lot for

the BBQ, Eurovision song contest and Christmas fika! It was always fun and

delicious! Björn, thanks for all the great talks in the lab and on the way home to

Flogsta! Carla thank you for great company in and outside the lab! Thanks for

the great Indian dinner at your place! Thanks Gucci, for the fikas, the glasses of

wine and chats! Elin E, thank you for your help with administrative stuff and of

course for the nice climbing sessions! Christina, thanks for keeping the lab so

organized and for always knowing where I could find stuff! Liza, for sharing

office space! Tonge, your good mood is contagious! Thanks! Agata and

Caroline, together with Karin, Linda and Elin L, thanks for the great time in

Berlin! It was a lot of fun! Junhong, thanks for the common courses we took

and organizing the Rudbeck masquerade together! Johan O, thanks for all the

computer help! Erik, thanks for all administrative work making it even easy to

fill out EU time sheets! Rasel, for always being happy and for fun talks!

Rachel, for being a great roommate at the retreat! Spyros, for your

43

encouragements and talks! Monica, Lena, Maria, Ida, Irene, Gaëlle, Felipe,

Andries, Lei, Anne-Li, Axel, Johanna and Joakim, thanks for great

discussions and making Rudbeck/BMC such a nice place!

For the people at SciLife: Thanks Tim, Elin B and Burcu for the nice

conversations over a beer, during collaborations or fika! Tim, thanks for the

tennis matches!

For Q-linea: Janne, thank you for your help with the Aquila in person and

electronically from far away. Thanks for fixing it every single time and giving

us an introduction about its inner life! Jenny, Anna and Jonas, thank you for

your help with the old blob counter and always welcoming us at Q-linea!

For my friends near and far: Anja, Franzi, Marlen, and Kasia, thank you for

the great friendship and support you have given me throughout the years! For

everything we experienced in the last couple of years, the talks, laughter, travels,

dinners and much more. It feels good to know that I can always count on you!

You made Sweden a much brighter place! Thanks Anja, Franzi and Marlen for

always offering me a place to stay in Uppsala! Merci, Kasia for hosting us in

Paris! It was great fun! Rachel, thanks for all the fun we had in Uppsala and

thank you for proof reading this thesis! Tina, thanks for being such a good

friend for all these years! For always lending me an ear and giving me support

when I needed it! For sharing the good and bad moments in life and always

having advice concerning work, life, just everything! No matter where you

lived, your door was always open for me! Thanks! Anja S, thanks for

encouragements, support and inspiration! Thank you for all the visits during the

last couple of years! Each one was unique and great! Anna, Alex K, and Anke,

vielen Dank für Eure Freundschaft! Ich bin mir sicher, dass egal wo es mich hin

verschlägt, ich mich auf Euch immer verlassen kann!

Micke, tack för allt vi har upplevt tillsammans under de gångna sex åren. Du har

alltid funnits där för mig när jag behövt dig som mest! Ord kan inte tillräckligt

beskriva hur tacksam jag är för det.

For my family: Mama, Papa, Oma, Opa und Omi, ich möchte mich von

Herzen bei Euch bedanken! Ihr habt mir mein Studium ermöglicht und ich

verdanke es Euch, dass ich bis hierhin gekommen bin. Nie habt ihr meine

Entscheidung oder mein Vermögen zu promovieren angezweifelt und ich konnte

Eurer Unterstützung immer sicher sein! Vielen Dank! Jens und Lars, ich kann

mich glücklich schätzen Euch als Brüder zu haben! Egal ob ich Hilfe mit

Übersetzungen, formellen Schreiben oder mit Weihnachtsgeschenken benötigte,

ich konnte immer auf Euch zählen. Danke!

44

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